Responses of Well-Differentiated Airway Epithelial Cell Cultures from Healthy Donors and Patients with Cystic Fibrosis to Burkholderia cenocepacia Infection

Abstract

Well-differentiated cultures established from airway epithelia of patients with cystic fibrosis (CF cultures) exhibited goblet cell hyperplasia, increased secretion of mucus, and higher basal levels of interleukin-8 than similarly cultured cells from healthy donors. Upon apical infection with low doses (10 4 to 10 5 CFU) of Burkholderia cenocepacia isolate BC7, the two cultures gave different responses. While normal cultures trapped the added bacteria in the mucus layer, killed and/or inhibited bacterial replication, and prevented bacterial invasion of the cells, CF cultures failed to kill and/or supported the growth of bacteria, leading to invasion of underlying epithelial cells, compromised transepithelial permeability, and cell damage. Depletion of the surface mucus layer prior to bacterial infection rendered the normal cultures susceptible to bacterial invasion, but the invading bacteria were mainly confined to vacuoles within the cells and appeared to be nonviable. In contrast, bacteria that invaded cells in CF cultures were found free in the cytoplasm surrounded by intermediate filaments and also between cells. Cultured CF airway epithelium was therefore more susceptible to infection than normal epithelium. This mimics CF tissue in vivo and illustrates differences in the way epithelia in CF patients and normal subjects handle bacterial infection. In addition, we found that the CF and normal cell cultures responded differently not only to isolate BC7 but also to isolates of other B. cepacia complex species. We therefore conclude that this cell culture model is suitable for investigation of B. cepacia complex pathogenesis in CF patients.

Rhinovirus Activates Interleukin-8 Expression via a Src/p110β Phosphatidylinositol 3-Kinase/Akt Pathway in Human Airway Epithelial Cells

“…Human primary airway epithelial cells obtained from the tracheal trimmings of donor lungs at the time of double lung transplantation were cultured in collagencoated plates with bronchial epithelial cell culture media (Cambrex, East Rutherford, NJ) as previously described (21,30). First-passage cells were either grown under submerged conditions or, for selected experiments, differentiated to a mucociliary phenotype by seeding on collagen-coated transwells.…”

Rhinovirus Disrupts the Barrier Function of Polarized Airway Epithelial Cells

“…Human airway epithelial cells, obtained from tracheal trimmings of anonymous donor lungs at transplantation, were grown at air-liquid interface for mucociliary differentiation, as previously described (24). Use of donor lungs was reviewed by the Institutional Review Board of the University of Michigan.…”

“…16HBE14o-or Calu-3 cells were infected with RV39 at a multiplicity of infection (MOI) of 1 for 24 hours and paracellular permeability of FITCinulin (1 mg/ml) was determined (24,26). In selected experiments, cultures were infected with bacteria and transmigration assessed by colony counting.…”

“…Differentiated cell cultures were infected apically with RV39 or RV1B (MOI of 1) or equivalent volume of sham, and incubated for 24 hours. NTHi (isolate 6P5H) were then added to the apical surface and incubated for another 24 hours. An aliquot of basolateral medium was plated to determine the number of bacteria transmigrating across the epithelium.…”

Use of Suppression-Subtractive Hybridization To Identify Genes in the Burkholderia cepacia Complex That Are Unique to Burkholderia cenocepacia

“…Invasion and survival in epithelial cells (4,7,56), macrophages (42), amoebae (41), and acanthamoeba (33) have also been demonstrated. Analysis of a signature-tagged mutagenesis (STM) library in a chronic pulmonary infection model identified several B. cenocepacia genes directly involved in host survival, including genes involved in cellular metabolism, regulation, DNA replication and repair, cell surface proteins, and polysaccharide production (31).…”